1. Field of the Invention
The present invention relates to the field of oligo- and polynucleotides, more particularly to modified oligo- and polynucleotides suitable for therapeutic and imaging purposes, including those which require the delivery of such oligo- and polynucleotides either into cells or to cell surfaces.
2. Brief Description of the Prior Art
Oligonucleotides and oligonucleotide analogs which are complementary to messenger RNAs encoded by human, animal, plant, microorganism and viral genomes have been shown to be effective for inhibiting or otherwise regulating gene expression, such as by hybrid arrest of translation. This has prompted several groups of workers to attempt to develop various types of therapeutic “antisense” oligo- and polynucleotides. For a recent review, see van der Krol, et al., Modulation of Eucaryotic Gene Expression by Complementary RNA or DNA Sequences, Biotechniques, 6:958-976 (1988).
One mechanism by which phosphodiester oligodeoxynucleotides have been found to promote hybrid arrest of translation is through RNase H cleavage of the RNA in an RNA:oligodeoxynucleotide duplex. See, Minshull and Hunt, The Use of Single-Stranded DNA and RNase H to Promote Quantitative Hybrid Arrest of Translation of mRNA/DNA Hybrids in Reticulocyte Lysate Cell-Free Translations, Nucleic Acids Research, 14:6433-6451 (1986); Cazenave, et al., Enzymatic Amplification of Translation Inhibition of Rabbit Beta-Globin mRNA Mediated by Anti-Messenger Oligodeoxynucleotides Covalently Linked to Intercalating Agents, Nucleic Acids Research, 15:4717-4736 (1987); and Dash, et al., Selective Elimination of mRNAs In Vivo: Complementary Oligodeoxynucleotides Promote RNA Degradation by an RNase H-Like Activity, Proc. Nat. Acad. Sci. USA, 84:7896-7900 (1987).
In view of this, sensitivity to RNase H is one of the properties that should be considered when developing effective therapeutic antisense oligonucleotides. Unmodified oligonucleotides with phosphodiester linkages can complex with RNA to form RNase H substrates but are not nuclease resistant. One method of achieving a high degree of efficiency of translation inhibition is to chemically alter the oligodeoxynucleotide in order to facilitate its entry into the cell and increase its half-life, while maintaining its affinity for the specific RNA of interest. Thus, other aspects of this undertaking would include the development of antisense oligonucleotides or polynucleotides with modifications that result in reduced sensitivity of these agents to nucleases which might diminish their effectiveness or render them inactive.
Inoue, et al., Sequence-Dependent Hydrolysis of RNA using Modified Oligodeoxynucleotide Splints and RNase H, Nucleic Acids Symposium Series, 18:221-224 (1987) describes a method of cleaving RNA in vitro in a site-specific manner using modified oligonucleotides and RNase H.
Partially or completely substituted phosphorothioates have also been tested for antisense function. See Marcus-Sekura, et al., Comparative Inhibition of Chloramphenicol Acetyltransferase Gene Expression by Antisense Oligonucleotide Analogues having Alkyl Phosphotriester, Methylphosphonate and Phosphorothioate Linkages, Nucleic Acids Research, 15:5749-5763 (1987) as well as Agrawal, et al., Oligodeoxynucleotide Phosphoramidates and Phosphorothioates as Inhibitors of Human Immunodeficiency Virus, Proc. Nat. Acad. Sci. USA, 85:7079-7083 (1988). They exhibit varying degrees of resistance to a variety of nucleases. Fully substituted phosphorothioate oligodeoxynucleotides form RNase H-sensitive hybrids, as reported in Stein, et al., Physicochemical Properties of Phosphorothioate Oligodeoxynucleotides, Nucleic Acids Research, 16:3209-3221 (1988).
By contrast, many oligonucleotides containing fully-substituted nuclease-resistant sugar-phosphate backbones are incapable of forming RNase H-sensitive hybrids with target DNAs. See, for example, Sun, et al., Sequence-Targeted Cleavage of Nucleic Acids by Oligo-alpha-thymidylate-Phenanthroline Conjugates: Parallel and Antiparallel Double Helices are formed with DNA and RNA, Respectively, Biochemistry, 27:6039-6045 (1988). Alpha-phosphodiesters form antiparallel helices with complementary RNA, but not with DNA. Nevertheless, although the alpha-linkages are nuclease-resistant, alpha DNA-RNA hybrids are not substrates for RNase H. Aminophosphonates or triesters can be formed by modifying the oxidation step in hydrogen-phosphonate synthesis. These uncharged linkages should interfere with the ability to form an RNase H-sensitive hybrid.
Oligodeoxynucleotide analogs containing methylphosponate linkages have been shown to have an antisense effect in vitro. See, Miller, et al., Control of Ribonucleic Acid Function by Oligonucleoside Methylphosphonates, Biochimie, 67:769-776 (1985) as well as Maher and Dolnick, Comparative Hybrid Arrest by Tandem Antisense Oligodeoxyribonucleotides or Oligodeoxyribonucleoside Methylphosphonates in a Cell-Free System, Nucleic Acids Research, 16:3341-3358 (1988). An antisense effect has been shown in vivo. See, Sarin, et al., Inhibition of Acquired Immunodeficiency Syndrome Virus by Oligodeoxynucleoside Methylphosphonates, Proc. Nat. Acad. Sci. USA, 85:7448-7451 (1988); Smith, et al., Antiviral Effect of an Oligodeoxynucleoside Methylphosphonate Complementary to the Splice Junction of Herpes Simplex Virus Type 1 Immediate Early Pre-mRNAs 4 and 5, Proc. Nat. Acad. Sci. USA, 83:2787-2791 (1986); and Agris, et al., Inhibition of Vesicular Stomatitis Virus Protein Synthesis and Infection by Sequence Specific Oligodeoxynucleoside Methylphosphonate, Biochemistry, 25:6268-6275 (1986). Methylphosphonamidite nucleosides can be incorporated during phosphoramidite synthesis to yield partially substituted structures, as reported by Marcus-Sekuar, supra. Reducing the number of methylphosphonate linkages leads to greater hybrid stability, as reported by Quartin and Wetmur, Effect of Ionic Strength on the Hybridization of Oligodeoxynucleotides with Reduced Charge Due to Methylphosphonate Linkages to Unmodified Oligodeoxynucleotides Containing the Complementary Sequence, Biochemistry, 28:1040-1047 (1989).
In a cell-free translation system, it was determined that the action of RNase H was not involved in the antisense effect seen with fully methylphosphonate-substituted oligodeoxynucleotides, and that such compounds did not form RNase H-sensitive hybrids with complementary RNA. See, Maher and Dolnick, supra.
Oligodeoxynucleotides with modified ends have been shown to have relative resistance to exonucleases. For example, see Agrawal and Goodchild, Oligodeoxynucleotide Methylphosphonates: Synthesis and Enzymic Degradation, Tetrahedron letters, 28:3539-3542 (1987).
Notwithstanding the progress made and reported as described above, these efforts have not been entirely successful in permitting the rational design of stable, therapeutically-effective oligo- and polynucleotides such as has now become possible as a result of the present invention. Particularly, no one has examined the ability of mixed oligodeoxynucleotides to form RNase H-sensitive substrates as a factor in optimizing antisense function. In addition, heretofore, no one has correlated the ability to form RNase H sensitive substrates with nuclease resistance.